PROCESS FOR THE PRODUCTION OF L-LYSINE USING CORINEFORM BACTERIA
Field of the Invention The invention provides a process for the production of L-lysine using corineforrae bacteria, which are sensitive to diaminopimelic acid analogs, in particular, to 4-hydroxy-diaminopimelic acid. Background of the Invention L-amino acids, in particular L-lysine, are used in human medicine, in the pharmaceutical industry, in the food industry, and more particularly in animal nutrition. The production of amino acids is known by the fermentation of strains of coryneform bacteria, in particular, corynebacterium glutamicum. Taking into account its great importance, constant efforts are made to improve production processes. Process improvements may relate to the measures employed in fermentation technology, such as, for example, agitation and oxygen supply, or the composition of the nutrient medium such as, for example, the concentration of sugar during fermentation, or the preparation to form the product by, for example, ion exchange chromatography or the intrinsic performance properties of the same microorganism.
Ref. -.160251 To improve the performance properties of these microorganisms, they are used. methods that involve mutagenesis, selection and choice of mutants. In this form, strains are obtained which are sensitive to antimetabolites such as, for example, the analogues of the S- (2-aminoethyl) -cysteine Usin, or which are auxotropic to regulate important metabolites that produce L-amino acids. For some years, the methods using recombinant DNA technology have been used to improve the strains that produce L-amino acids of Corynebacterium glutamicum, amplifying the genes of the biosynthesis of the individual amino acid and investigating the effect on the production of the L-amino acid. Brief Description of the Invention The inventors have been involved in the invention of new principles to improve the processes of the fermentative production of L-lysine using the bacterium corineforrae. Detailed Description of the Invention When referred to hereinafter as L-lysine or lysine, it is understood that not only the bases are included, but also salts such as, for example, lysine monohydrochloride or lysine sulfate. The invention provides a process for the fermentative production of L-lysine using a coryneform bacterium that is resistant to diaminopimelic acid analogs, in particular, 4-hydroxy-diaminopimelic acid. Analogs are generally used in concentrations of > (greater than / equal to) 3 a (less than / equal to) 30 g / 1. The invention also provides a process for the fermentative production of L-lysine using coryneform bacteria which already produce L-lysine, and which are resistant to diaminopimelic acid analogues, in particular 4-idroxydiaminopimelic acid. This invention further provides a process for the production of L-lysine in which the following steps are carried out: a) fermentation of the coryneform bacteria that produce L-lysine, which are at least sensitive to the diaminopimelic acid analogs, in particular, 4-hydroxy diaminopimelic acid. b) enrichment of L-lysine in the medium or in the bacterial cells; and optionally c) isolation of L-lysine or food additives containing L-lysine from a fermentation broth, so that it is > 0 to 100% of the constituents from the fermentation broth and / or from the biomass that is present. The invention similarly provides a process for the production of coryneform bacteria that are resistant to diaminopimelic acid analogs, in particular, 4-hydroxy-diaminopimelic acid. The strains that are used produce L-lysine, preferably before resisting 4-hydroxy-diaminopimelic acid. The expression of the diaminopimelic acid analogs according to the present invention includes compounds such as 4-fluorodiaminopimelic acid, 4-hydroxy diaminopimelic acid, 4-oxodiaminopimelic acid, or 2, 4, 6-triaminopimelic acid. The present invention also provides mutant coryneform bacteria that produce L-lysine which are resistant to one or more of the diaminopimelic acid analogs selected from the group comprising 4-fluoro-diaminopimelic acid, 4-hydroxy-diaminopimelic acid, 4-oxodiaminopimelic acid or 2, 4-acid. 6-triaminopimelic. The invention also provides food additives based on a fermentation broth containing L-lysine produced according to the invention and without or only traces of biomass and / or constituents from the fermentation broth formed during the fermentation of the microorganisms that produce L-lysine. It is understood that the term "traces" means quantities of >; 0% to 5%.The invention further provides food additives based on the fermentation broth, characterized in that a) they contain L-lysine produced according to the invention, and b) they contain the biomass and / or constituents of the fermentation broth in an amount of 90% a 100% that is formed during the fermentation of microorganisms that produce L-lysine. The microorganisms that are provided from the present invention can produce amino acids of glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These microorganisms may be representative of the coryneform bacteria, in particular of the genus Corynebacterium. Among the genus Corynebacterium, mention should be made in particular of the species Corynebacterium glutamicum, which is known to those skilled in the art for its ability to produce L-amino acids. Suitable strains of the genus Corynebacterium, in particular the species Corynebacterium glutamicum, are in particular the following strains of wild type known Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and mutants that produce L-amino acids and / or strains produced by them, such as, for example, the strains that produce L-lysine Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 Corynebacterium glutamicum ATCC 21513 Corynebacterium glutamicum ATCC 21544 Corynebacterium glutamicum ATCC 21543 Corynebacterium glutamicum DSM 4697 and Coryn ebacterium glutamicum DSM 5715. It has been found that the coryneform bacterium that is sensitive to diaminopimelic acid analogues, in particular to 4-hydroxy-diaminopimelic acid, produce L-lysine in an improved form. In order to produce the coryneform bacterium according to the invention which is sensitive to 4-hydroxy-diaminopimelic acid, mutagenesis methods described in the prior art are used. For mutagenesis, conventional in vivo mutagenesis processes can be employed using mutagenic substances such as, for example, N-methyl-N '-nitro-N-nitrosoguanidine or ultraviolet light (Miller, JH: A Short Course in Bacterial Genetics. Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992). The coryneform bacterium that is sensitive to 4-hydroxy diaminopimelic acid can be identified by plating with a nutrient medium containing 4-hydroxy-diaminopimelic acid. The final concentrations of ca. 5 to 15 g / 1, for example, ca. 10 g / 1 of 4-hydroxy-diamino-pimellic acid in the nutrient medium are particularly suitable for this purpose. At this concentration, mutants sensitive to 4-hydroxy diaminopimelic acid can be distinguished from the unchanged precursor strains by late growth. After selection, mutants to 4-Mdroxydiaminopimelic acid exhibit improved L-lysine production. Furthermore, it may be advantageous for the production of L-lysine, in addition to the sensitivity to 4-hydroxy-diaminopimelic acid, to improve, in particular, overexpress, one or more enzymes of the respective biosynthesis path, glycolysis, anaplerosis, citric acid cycle, Pentose phosphate cycle, export of amino acids and optionally, regulatory proteins. In general, the use of genes is preferred.
It is understood that the terms "endogenous genes" or "endogenous nucleotide sequences" mean the genes or nucleotide sequences present in the population of a species. The expressions "improve" and "to improve" describe in this context, the increase of the intracellular activity of one or more enzymes or proteins in a microorganism that is encoded by the corresponding DNA, through for example, the increase in the number of copies of the gene or genes, which employ a strong promoter or a gene that encodes a corresponding enzyme or protein having a high activity and, optionally, combining these measurements. By means of this improvement, in particular the overexpression measures, the activity or concentration of the corresponding protein is generally raised at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, referred to the activity or concentration of the wild-type protein and / or the activity or concentration of the protein in the starting microorganisms. Thus, for the production of L-lysine, in addition to the sensitivity to diaminopimelic acid analogues, in particular 4-droxidiaminopimelic acid, one or more of the genes selected from the following group can be improved, in particular overexpressed: the lysC gene which encodes a feedback-resistant aspartate kinase (Accession No. P26512, EP-B-0387527, EP-A-099999, 0000638388), The dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197 335 ), the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992) .. Journal of Bacteriology 174: 6076-6086), simultaneously, the pyc gene encoding pyruvate carboxylase (DE-A-198 31 609, EP-A-1108790), the zwf gene encoding glucose-6-phosphate dehydrogenase (JP-A-09224661, EP-A-1108790), simultaneously, the lysE gene encoding the protein exporting lysine (DE-A-195 48 222), the zwal gene encoding the Zwal protein (DE : 19959328.0, DS 13115), the lysA gene coding for diaminopimelic acid decarboxylase (No. access X07563), the sigC gene coding for sigma factor C (DE: 10043332.4, DSM14375), the tpi gene coding for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086) and the pgk gene coding for 3-phosphoglycerate kinase
(Eikmanns (1992), Journal of Bacteriology 174: 6076-6086). Furthermore, it may be advantageous for the production of L-lysine, in addition to the sensitivity of 4-hydroxy: oxydiamine pimelic acid, to simultaneously attenuate, in particular reduce the expression, of one or more of the genes selected from the following group: the pck gene coding for phosphoenol pyruvate carboxy kinase (DE 199 50 409.1, DSM 13047), the pgi gene coding for glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969), the poxB gene coding for the pyridate oxidase
(DE: 1995 1975.7, DSM 13114), the deaD gene encoding DNA helicase (DE: 10047865.4, DSM14464), the citE gene coding for citrate lyase (PCT / EP01 / 00797, DSM13981), the menE gene encoding for O-succinylbenzoic acid CoA-ligase (DE: 10046624.9, DSM14080), the mikE17 gene encoding mikE17 transcription regulator (DE: 10047867.0, DSM14143) and the zwa2 gene encoding the Zwa2 protein (DE:
19959327. 2, DSM 13113). The term "attenuation" describes in this connection, the reduction or extinction of the intracellular activity of one or more enzymes (proteins) in a microorganism that is encoded by the corresponding DNA, using for example, a weak promoter or a gene or allele that encodes a corresponding enzyme with reduced or inactive activity to the corresponding gene or enzyme (protein), and optionally combine these measurements. By means of these attenuation measurements, the activity or concentration of the corresponding protein is generally reduced from 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10% 6 from 0 to 5% of the activity or concentration of the wild-type protein, and / or the activity or concentration of the protein in the initial microorganism. Finally, it may also be advantageous for the production of L-lysine, in addition to the sensitivity to 4-hydroxy-diaminopimelic acid, to quench the undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academia Press, London, UK, 1982). The microorganisms produced according to the invention are also encompassed in the invention and can be grown continuously or discontinuously in a batch process (batch culture) or in a feedback process (feeding process) or repeated feedback process (feeding process) repetitive) for the purpose of producing L-lysine. A summary of the known cultivation methods is described in the textbook of Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (Bioreaktoren und peiphere Einrichtungen (Vieweg Verlag, Brunswick / Wiesbaden, 1994)). The culture medium to be used must adequately satisfy the requirements of the respective strains. Descriptions of the culture medium for various microorganisms are contained in the "Manual of Methods for General Bacteriology" of the American Bacteriology Society (Washington DC, USA, 1981). As a carbon source, sugars and carbohydrates can be used such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, for example, soybean oil, sunflower oil, oil of peanut and coconut oil, fatty acids such as for example, palmitic acid, stearic acid and linoleic acid, alcohols such as for example, glycerol and ethanol and organic acids such as for example, acetic acid. These substances can be used individually or as a mixture. As a nitrogen source, organic nitrogen containing compounds such as peptones, yeast extract, meat extract, malt extract, soluble corn extract, soy bean flour and urea, inorganic compounds such as ammonium sulfate, can be used. ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually as a mixture. As the phosphorus source, phosphoric acid, potassium acid phosphate or dipotassium acid phosphate or the corresponding sodium-containing salts can be used. The culture medium must also contain metal salts such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth promoters such as amino acids and vitamins can also be used for the aforementioned substances. Apart from these, suitable precursors can be added to the culture medium. The aforementioned starting substances can be added to the culture in the form of a single batch or can be fed in an appropriate manner during cultivation. In order to regulate the pH of the basic compounds of the culture, sodium hydroxide, potassium hydroxide, ammonia or water of ammonia or acidic compounds such as phosphoric acid or sulfuric acid are used as appropriate. To control foaming, antifoaming agents such as, for example, fatty acid polyglycol esters can be used. To maintain the stability of the plasmids, substances that selectively act, for example, antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or gas mixtures containing oxygen are fed into the culture. The culture temperature is usually from 20 ° C to 45 ° C, and preferably at 25 ° C to 40 ° C. The cultivation is continued until the maximum amount of the desired product has been formed. This goal is usually achieved within 10 to 160 hours. Methods for the determination of L-lysine are known from the prior art. The analysis can be carried out as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography followed by ninhydrin derivatization, or by reverse phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174). The process according to the invention serves for the fermentative production of L-lysine. The concentration of L-lysine can optionally be adjusted to the desired value by the addition of L-lysine. By the processes described, it is possible to isolate coryneform bacteria which are sensitive to diaminopimelic acid analogs, in particular 4-hydroxy-diaminopimelic acid and to produce L-lysine in an improved form, according to the fermentation processes described. Example 1 Separation by exclusion of mutants sensitive to 4-hydroxy diaminopimelic acid. The strain Corynebacterium glutamicum DSM13994 was produced by the non-directed mutagenesis, selection and mutant selection of C. glutamicum ATCC13032. Strain DSM13994 is sensitive to the lysine analog S- (2-aminoethyl) -Listein and has an aspartate kinase that is insensitive to feedback to inhibition by mixtures of lysine (or the lysine analog S- (2-aminoethyl) - L-cysteine, 100 mM) and threonine (10 mM), while in contrast to this, the activity of aspartate kinase in the wild type is inhibited in a residual activity up to 10%. A pure culture of strain DSM13994 was deposited on January 16, 2001 in the German Collection for Microorganisms and cell cultures (DSM Brunswick) according to the Budapest Convention. For separation by exclusion in colonies that are sensitive to 4-hydroxy-diaminopimelic acid, strain DSM 13994 after UV mutagenesis (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, New York, 1989 ) is placed on LB agar plates containing 4-hydroxy diaminopimelic acid. The agar plates are supplied with 10 g / 1 of 4-hydroxy diaminopimelic acid. The growth of the colonies is observed for 48 hours. At this concentration, the matants that are sensitive to 4-M-roxydiaminopimelic acid can be differentiated from the unchanged precursor strain by improved growth. In this way, a clone showing a substantially delayed growth to the DSM 1399 is identified. The strain is identified as DSM 13994_Hdap_s. Example 2 Production of lysine Strain C. glutamicum DSM 13994_Hdap_s obtained in example 1 is cultured in a nutrient medium suitable for lysine production and the lysine content in the culture supernatant is determined.
For this purpose, all strains are first incubated on agar plates for 24 hours at 33 ° C. Using this culture on agar plates, the preculture is inoculated (10 ml of the medium in a 100 ml Erlenmeyer flask). The MM medium is used as a means for preculture. The preculture is incubated for 24 hours at 33 ° C at 240 rpm in a vibrator. Using this preculture, the main culture is inoculated so that the initial optical density (OD- 660 nm) of the main culture is 0.1 OD. The MM medium is also used for the main culture. Medium MM
The CSL (fermented corn liquor), MOPS (morpholinopropanesulfonic acid) and the saline were adjusted with water and ammonia to pH 7 and autoclaved. The sterile substrate and the vitamin solutions as well as the dry autoclaved CaC03 were added.
The culture was carried out in a 100 ml Erlenmeyer flask with a volume of 10 ml equipped with bulkheads. The cultivation is carried out at 33 ° C and 80% atmospheric humidity. After 72 hours, the OD is determined by measuring the wavelength of 660 nm with a Biomek 1000 instrument (Beckmann Instruments GmbH, Munich). The amount of the lysine formed is determined by ion exchange chromatography and post-column derivatization with ninhydrin detection using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany). The result of the experiment is shown in Table 1 Table 1
It is noted that in relation to this date, the best known method for carrying out the aforementioned invention is that which is clear from the present description of the invention.